A proposed role for the matrix (MA) domain of the HIV-1 Gag protein is to facilitate the incorporation of HIV-1 envelope glycoproteins into virus particles. To characterize functions of HIV-1 MA, including envelope incorporation into virus particles, we introduced over 65 single and double amino acid substitution mutations throughout HIV-1 MA. We demonstrated that single amino acid substitutions in MA residues 12 or 30 blocked the virion incorporation of HIV-1 envelope glycoproteins, and that this block could be reversed by pseudotyping with heterologous retroviral envelope glycoproteins containing short cytoplasmic tails, or by truncating 104 or 144 amino acids from the cytoplasmic tail of the HIV-1 transmembrane glycoprotein gp41. To map the domain of the gp41 cytoplasmic tail responsible for the block to virion incorporation imposed by the MA mutations, a series of eight additional truncation mutations were constructed between 23 and 93 amino acids from the C- terminus of gp41. The data indicated that virion incorporation of HIV-1 envelope glycoproteins with truncations of 23, 30, 51, and 56 amino acids from the C-terminus of gp41 is specifically blocked by the MA amino acid 12 mutation, whereas truncations of greater than 93 amino acids reverse the envelope incorporation defect. These results suggest that residues within a predicted `-helix located between 63 and 87 amino acids from the gp41 C-terminus may interact with MA to facilitate the incorporation of envelope glycoproteins with long cytoplasmic tails into HIV-1 virions. To obtain more information about the role of MA and envelope incorporation, we obtained and analyzed viral revertants of two MA residue 12 mutations. Nucleotide sequencing of the revertants indicated that a Val->Ile substitution at MA amino acid 34 compensated for both of the amino acid 12 mutations, providing support for an interaction between residue 12 and 34 during the envelope incorporation process.